Immunomodulatory methods using oligosaccharides

ABSTRACT

Methods for modulating immune responses are provided. The methods involve contacting an immune cell with an agent that modulates interaction of a compound comprising a Lewis antigen with the immune cell such that production by the immune cell of at least one cytokine that regulates development of a T helper type 1 or T helper type 2 response is modulated. In one embodiment, the agent is a stimulatory form of a compound comprising a Lewis antigen, such as a Lewis y , Lewis x  or Lewis a  oligosaccharide, or a derivative thereof. In another embodiment, the agent is an inhibitory form of a compound comprising a Lewis antigen, such as a Lewis y , Lewis x  or Lewis a  oligosaccharide, or a derivative thereof. In various embodiments, the immune cell is a human immune cell, a macrophage or a T cell. Pharmaceutical compositions for modulating immune responses are also provided.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/033984 filed Jan. 11, 2005, which is a continuation of U.S.application Ser. No. 08/597518 filed Jan. 31, 1996 (now U.S. Pat. No.6,841,543, issued Jan. 11, 2005), the entire contents of which arehereby incorporated by reference.

GOVERNMENT FUNDING

Work described herein was supported under grant AI 27448 awarded by theNational Institutes of Health. The U.S. government therefore may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

The T lymphocyte compartment of the immune system can be divided into avariety of cell subsets. For example, CD4⁺ T cells represent the Thelper cell subset, whereas CD8⁺ T cells represent the cytotoxic T cellsubset. Additionally, CD4⁺ T helper cells mature into distinctsubpopulations that produce different panels of cytokines: the T helpertype 1 (Th1) subset produces interleukin-2 (IL-2), interferon-γ (IFN-γ)and tumor necrosis factor-β (TNF-β), whereas the T helper type 2 (Th2)subset produces interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6) and interleukin-10 (IL-10). The Th1 and Th2 subsetsalso have differing functional activities. Th1 cells are involved ininducing delayed type hypersensitivity responses, whereas Th2 cells areinvolved in providing efficient “help” to B lymphocytes and stimulateproduction of IgG1 and IgE antibodies. For a review of Th1 and Th2subsets, see Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol.12:635 -673.

Cytokines are thought to play a dominant role in controlling thedifferentiation of T helper precursors (Thp) to either the Th1 or Th2lineage. Th1-associated cytokines, such as IFN-γ, can enhance thedevelopment of Th1 cells and inhibit the development of Th2 cells,whereas Th2-associated cytokines, such as IL-4 and IL-10, can enhancethe development of Th2 cells and inhibit the development of Th1 cells.Thus, cytokines can reciprocally regulate the development and/orprogression of either a Th1 or a Th2 response.

The course of certain disease states is influenced by whether apredominant Th1 response or Th2 response is mounted. For example, inexperimental leishmania infections in mice, animals that are resistantto infection mount predominantly a Th1 response, whereas animals thatare susceptible to progressive infection mount predominantly a Th2response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72;Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is observedcoincident with the release of eggs into the tissues by female parasitesand is associated with a worsening of the disease condition (Pearce, E.J., et al. (1991) J. Exp. Med. 173:159-166; Grzych, J-M., et al. (1991)J. Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.148:3264-3270). Many human diseases, including chronic infections (suchas with human immunodeficiency virus (HIV) or tuberculosis) and certainmetastatic carcinomas, also are characterized by a Th1 to Th2 switch,with elevated expression of IL-10 (see e.g., Shearer, G. M. and Clerici,M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M.(1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J. Clin.Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA89:7708-7712; Fauci, A. S. (1988) Science 239:617-623). Furthermore,certain autoimmune diseases have been shown to be associated with apredominant Th1 response. For example, patients with rheumatoidarthritis have predominantly Th1 cells in synovial tissue (Simon, A. K.,et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimentalautoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1cells (Kuchroo, V. K., et al. (1993) J. Immunol. 151:4371-4381).

Velupillai and Ham (Proc. Natl. Acad. Sci. USA (1994) 91:18-22) haveshown that schistosome egg antigen (SEA), which expresses the Lewis^(x)antigen, and conjugates of the Lewis^(x) antigen, can stimulate IL-10production by B cells from Schistosoma mansoni infected mice, but not Bcells from uninfected mice, suggesting that during the course of S.mansoni infection, the observed Th1 to Th2 shift may results from IL-10production by B cells induced by SEA. This work, however, did notdemonstrate whether human immune cells (e.g., human immune cells in theabsence of S. mansoni infection) were responsive to Lewisantigen-containing compounds, nor whether cell types other than B cells,such as macrophages or T cells, could produce IL-10 in response tostimulation with compounds comprising a Lewis antigen in the absence ofS. mansoni infection. Moreover, this work did not demonstrate whetherproduction other cytokines that regulate development of Th1 and Th2responses, such as IL-4, could be stimulated

SUMMARY OF THE INVENTION

Given the role of either Th1 or Th2 cells in the development orprogression of many disease states, methods for influencing whether aTh1 or Th2 response is mounted are desirable for a variety of clinicalsituations. This invention provide methods for modulating immuneresponses by modulating the interaction of immune cells with a compoundcomprising a Lewis antigen such that production by the immune cells ofat least one cytokine that regulates development of a Th1 or Th2response is modulated. The invention is based, at least in part, on thediscovery that stimulation of human immune cells, T cells or macrophageswith Lewis antigen-containing conjugates results in the production ofcytokines that regulate the development of Th1 or a Th2 response.Moreover, it has now been discovered that human immune cells aresensitive to stimulation by Lewis^(y) antigen-containing conjugates,that cells from human allergy patients and cancer patients showresponsiveness to Lewis antigens, that IL-4 production can be stimulatedby Lewis antigen-containing conjugates and that conjugates wherein thesugars represent approximately 20-24% of the conjugate by weight, orgreater, are preferred for stimulation. The immunomodulatory methods ofthe invention allow for an immune response to be directed to either aTh1 or a Th2 response. The ability to influence the development ofeither a Th1 or a Th2 response using the immunomodulatory methods of theinvention is applicable to the treatment of a wide variety of disorders,including cancer, infectious diseases (e.g., HIV and tuberculosis),allergies and autoimmune diseases.

In one embodiment, the invention provides an immunomodulatory methodcomprising contacting a human immune cell with an agent that modulatesinteraction of a compound comprising a Lewis antigen with the humanimmune cell such that production by the human immune cell of at leastone cytokine that regulates development of a Th1 or Th2 response ismodulated. The human immune cell can be, for example, a T cell, amacrophage or a B cell. In another embodiment, the invention provides animmunomodulatory method comprising contacting a macrophage with an agentthat modulates interaction of a compound comprising a Lewis antigen withthe macrophage such that production by the macrophage of at least onecytokine that regulates development of a Th1 or Th2 response ismodulated. In yet another embodiment, the invention provides animmunomodulatory method comprising contacting a T cell with an agentthat modulates interaction of a compound comprising a Lewis antigen withthe T cell such that production by the T cell of at least one cytokinethat regulates development of a Th1 or Th2 response is modulated.

In one embodiment of the immunomodulatory methods of the invention,production by immune cells of at least one cytokine (preferably IL-10 orIL-4) that regulates development of a Th1 or Th2 response is stimulated.In this embodiment, the agent with which the immune cells are contactedpreferably is a stimulatory form of a compound comprising a Lewisantigen, such as a compound comprising cross-linked (i.e., multivalent)Lewis^(y) oligosaccharides, Lewis^(x) oligosaccharides, Lewis^(a)oligosaccharides or derivatives thereof (e.g., sulfated, sialylated orsulfo-sialylated forms of these oligosaccharides). The stimulatorycompound can be, for example, a conjugate of the Lewis antigen and acarrier molecule (e.g., human serum albumin or polyacrylamide). Forstimulating responses by human immune cells, the agent preferablycomprises a Lewis^(y) oligosaccharide or a derivative thereof.

In another embodiment of the immunomodulatory methods of the invention,production by immune cells of at least one cytokine (preferably IL-10 orIL-4) that regulates development of a Th1 or Th2 response is inhibited.In this embodiment, the agent with which the immune cells are contactedpreferably is an inhibitory form of a compound comprising a Lewisantigen, such as a soluble, monovalent (i.e., non-crosslinked) form of aLewis^(y) oligosaccharide, a Lewis^(x) oligosaccharide, a Lewis^(a)oligosaccharide or a derivative thereof (e.g., sulfated, sialylated orsulfo-sialylated forms of these oligosaccharides). For inhibitingresponses by human immune cells, the agent preferably comprises aLewis^(y) oligosaccharide or a derivative thereof.

The stimulatory or inhibitory compounds of the invention can becontacted with immune cells in vitro to produce one or more cytokinesthat regulate the development of a Th1 or Th2 response. After in vitrostimulation, the immune cells can be administered to a subject toinfluence whether a Th1 or a Th2 response predominates in the subject.Alternatively, a stimulatory or inhibitory compound of the invention canbe administered to a subject such that production of at least onecytokine that regulates development of a Th1 or Th2 response is eitherstimulated or inhibited, respectively, in the subject, therebyinfluencing whether a Th1 or a Th2 response predominates in the subject.Accordingly, another aspect of the invention pertains to pharmaceuticalcompositions suitable for pharmaceutical administration. Thepharmaceutical compositions of the invention typically comprise astimulatory or inhibitory agent of the invention (e.g., a compoundcomprising a Lewis antigen) and a pharmaceutically acceptable carrier.In one embodiment, the composition is formulated to modulate responsesby human immune cells. In this embodiment, the active agent preferablycomprises a Lewis^(y) oligosaccharide. In another embodiment, thecomposition is formulated to modulate responses by macrophages. In yetanother embodiment, the composition is formulated to modulate responsesby T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph depicting the proliferation of splenocytes fromS. mansoni infected mice when stimulated with either media alone, Con A,LPS. anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 1B is a graph depicting the proliferation of splenocytes from S.mansoni infected mice when stimulated with increasing amounts of eitherLewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

FIG. 1C is a bar graph depicting the proliferation of splenocytes fromuninfected mice when stimulated with either media alone, Con A, LPS.anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 1D is a graph depicting the proliferation of splenocytes fromuninfected mice when stimulated with increasing amounts of eitherLewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

FIG. 2A is a bar graph depicting the production of IL-10 by splenocytesfrom S. mansoni infected mice when stimulated with either media alone,Con A, LPS. anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 2B is a graph depicting the production of IL-10 by splenocytes fromS. mansoni infected mice when stimulated with increasing amounts ofeither Lewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

FIG. 2C is a bar graph depicting the production of IL-10 by splenocytesfrom uninfected mice when stimulated with either media alone, Con A,LPS. anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 2D is a graph depicting the production of IL-10 by splenocytes fromuninfected mice when stimulated with increasing amounts of eitherLewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

FIG. 3A is a bar graph depicting the production of IL-4 by splenocytesfrom S. mansoni infected mice when stimulated with either media alone,Con A, LPS. anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 3B is a graph depicting the production of IL-4 by splenocytes fromS. mansoni infected mice when stimulated with increasing amounts ofeither Lewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

FIG. 3C is a bar graph depicting the production of IL-4 by splenocytesfrom uninfected mice when stimulated with either media alone, Con A,LPS. anti-IgM, SEA or LNFP III conjugated to HSA.

FIG. 3D is a graph depicting the production of IL-4 by splenocytes fromuninfected mice when stimulated with increasing amounts of eitherLewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x) antigen orsialyl-Lewis^(a) antigen, each conjugated to polyacrylamide.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides immunomodulatory methods in which a cell (e.g.,a human immune cell, a macrophage or a T cell) is contacted with anagent which modulates production by the cell of one or more cytokinesthat regulate Th1 or Th2 responses. The invention is based, at least inpart, on the discovery that when human immune cells, macrophages or Tcells are stimulated with a multivalent, crosslinked form of a Lewisantigen, the cells produce one or more cytokines (such as IL-10 and/orIL-4) that promote the development of a Th2 response and inhibit thedevelopment of a Th1 response. Human immune cells that exhibit thisresponsiveness include T cells from a pollen allergic individual andperipheral blood mononuclear cells from individuals with colon or lungcarcinomas or B lymphoma. Moreover, it has now been discovered thathuman immune cells preferentially respond to compounds comprising aLewis^(y) oligosaccharides. Still further, it has now been discoveredthat Lewis antigens conjugated to a carrier at a ligand density suchthat the sugars represent approximately 20-24% of the conjugate byweight are especially effective at stimulating production of cytokinesthat promote the development of a Th2 response (e.g., IL-10 and IL-4),an in particular can stimulate IL-4 production by splenocytes ofuninfected subjects. A preferred carrier for such stimulatory conjugatesis polyacrylamide.

In contrast to these stimulatory methods, contact of the cells with amonovalent, non-crosslinked form of the Lewis antigen inhibits theproduction of cytokines that promote Th2 responses and downregulate Th1responses that normally are produced when the cells encounter astimulatory form of a Lewis antigen. Thus, the methods of the inventionallow for cytokine production either to be stimulated or inhibited,depending on whether a stimulatory agent or an inhibitory agent is used.Accordingly, the immunomodulatory methods of the invention allow for animmune response to be directed to either a Th1 or a Th2 response. Theability to influence the development of either a Th1 or a Th2 responseusing the immunomodulatory methods of the invention is applicable to thetreatment of a wide variety of disorders, including cancer, infectiousdiseases (e.g., HIV and tuberculosis), allergies and autoimmunediseases.

In order that the present invention may be more readily understood,certain terms are first defined. Standard abbreviations for sugars areused herein.

As used herein, the term “Lewis antigen” is intended to includecarbohydrates having as a core sequence either the lacto type Istructure {Gal(β1-3)GlcNac} or the lacto type II structure{Gal(β1-4)GlcNac}, substituted with one or more fucosyl residues. TheLewis antigen may comprise a single substituted core sequence or arepetitive series of substituted core sequences. Moreover, the coresequence may be present within a larger sugar. Accordingly, a Lewisantigen-containing oligosaccharide can be, for example, a trisaccharide,a tetrasaccharide, a pentasaccharide, and so on. Types of Lewis antigensinclude Lewis^(x), Lewis^(y), Lewis^(a) and Lewis^(b) oligosaccharidesand derivatives thereof. Synthetic structural homologues of thesecarbohydrates that retain the immunomodulatory capacity described hereinare also intended to be encompassed by the term “Lewis antigen”.

As used herein, the term “Lewis^(x) oligosaccharide” refers to a lactotype II carbohydrate comprising the structure:{Gal(β1-4)[Fuc(α1-3)]GlcNac}.

As used herein, the term “Lewis^(y) oligosaccharide” refers to a lactotype II carbohydrate comprising the structure:{Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNac}.

As used herein, the term “Lewis^(a) oligosaccharide” refers to a lactotype I carbohydrate comprising the structure:{Gal(β1-3)[Fuc(α1-4)]GlcNac}.

As used herein, the term “Lewis^(b) oligosaccharide” refers to a lactotype I carbohydrate comprising the structure:{Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNac}.

As used herein, a “derivative” of a Lewis oligosaccharide refers to aLewis oligosaccharide having one or more additional substituent groups.Examples of derivatives include terminally sialylated forms of Lewisoligosaccharides (e.g., sialyl-Lewis^(x), sialyl-Lewis^(y),sialyl-Lewis^(a), sialyl-Lewis^(b)), sulfated forms of Lewisoligosaccharides and sulfo-sialylated forms of Lewis oligosaccharides.

As used herein, the term “human immune cell” is intended to includecells of the human immune cell which are capable of producing cytokinesthat regulate the development of a Th1 or Th2 response. Examples ofhuman immune cells include human T cells, human macrophages and human Bcells.

As used herein, the term “macrophage” is intended to include all cellswithin the macrophage lineage, including monocytes, circulatingmacrophages, tissue macrophages, activated macrophages, and the like,from a mammal (e.g., human or mouse).

As used herein, the term “T cell” (i.e., T lymphocyte) is intended toinclude all cells within the T cell lineage, including thymocytes,immature T cells, mature T cells and the like, from a mammal (e.g.,human or mouse).

As used herein, a “T helper type 2 response” (Th2 response) refers to aresponse by CD4⁺ T cells that is characterized by the production of oneor more cytokines selected from IL-4, IL-5, IL-6 and IL-10, and that isassociated with efficient B cell “help” provided by the Th2 cells (e.g.,enhanced IgG1 and/or IgE production).

As used herein, a “T helper type 1 response” (Th1 response) refers to aresponse by CD4⁺ T cells that is characterized by the production of oneor more cytokines selected from IL-2, IFN-g and TNF-β, and that isassociated with delayed type hypersensitivity responses.

As used herein, the term “a cytokine that regulates development of a Th1or Th2 response” is intended to include cytokines (produced by Th1 orTh2 cells or other cell types), that have a positive or negative effecton the initiation and/or progression of a Th1 or Th2 response, andespecially those cytokines that have reciprocal effects on thedevelopment of the Th1 vs. Th2 subpopulations, in particular, cytokinesthat promote the development of a Th2 response and downregulate thedevelopment of a Th1 response. Preferred cytokines that are produced bythe methods of the invention are IL-10, IL-4 and prostaglandin E₂(PGE₂). The most preferred cytokine produced by the methods of theinvention is IL-10.

As used herein, the term “development of a Th1 or Th2 response” isintended to include initiation of either a Th1 or Th2 response (e.g.,commitment of T helper precursors to either the Th1 or Th2 lineage) andprogression of either a Th1 or Th2 response (e.g., furtherdifferentiation of cells to either the Th1 or Th2 phenotype and/orcontinued function of Th1 or Th2 cells during an ongoing immuneresponse). Thus, a cytokine(s) produced in accordance with the methodsof the invention may have an effect on the initiation of a Th1 or Th2response, the progression of a Th1or Th2 response, or both.

As used herein, the various forms of the term “modulation” are intendedto include stimulation (e.g., increasing or upregulating a particularresponse or activity) and inhibition (e.g., decreasing or downregulatinga particular response or activity).

As used herein, the term “contacting” (i.e., contacting an agent with acell) is intended to include incubating the agent and the cell togetherin vitro (e.g., adding the agent to cells in culture) and administeringthe agent to a subject such that the agent and cells of the subject arecontacted in vivo.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Immunomodulatory Agents

In the immunomodulatory methods of the invention, a cell (e.g., a humanimmune cell, macrophage or T cell) is contacted with an agent thatmodulates interaction of a compound comprising a Lewis antigen with thecell such that production by the cell of at least one cytokine thatregulates development of a Th1 or Th2 response is modulated. Preferably,the agent itself comprises a Lewis antigen, as described in furtherdetail below. In one embodiment, the agent is a “stimulatory agent”,which stimulates production by the cell of at least one cytokine thatregulates development of a Th1 or Th2 response. In another embodiment,the agent is an “inhibitory agent”, which inhibits production by thecell of at least one cytokine that regulates development of a Th1 or Th2response (i.e., the inhibitory agent can block the production ofcytokines that normally occurs when the cell encounters a stimulatoryform of a Lewis antigen).

A. Stimulatory Agents

The stimulatory agents of the invention stimulate production by cells(e.g., human immune cells, macrophages or T cells) of at least onecytokine that regulates development of a Th1 or Th2 response. In apreferred embodiment, the stimulatory agent is a stimulatory form of acompound comprising a Lewis antigen. A “stimulatory form of a compoundcomprising a Lewis antigen” typically is one in which the carbohydratestructure is present in a multivalent, crosslinked form. In a preferredembodiment, the stimulatory form of a compound comprising a Lewisantigen is a conjugate of a carrier molecule and multiple carbohydratemolecules expressing a Lewis antigen. For example, carbohydratemolecules can be conjugated to a protein carrier, such as a conjugate ofhuman serum albumin (HSA) and Lewis^(y) oligosaccharides (referred toherein as HSA-Le^(y)). When a sugar-carrier protein conjugate is to beadministered to a subject, the carrier protein should be selected suchthat an immunological reaction to the carrier protein is not stimulatedin the subject (e.g., a human carrier protein should be used with ahuman subject, etc.). Alternative to a carrier protein, multiple Lewisantigens can be conjugated to other carrier molecules, such as a solidsupport, such as beads (e.g., polyacrylamide, agarose, sepharose,polystyrene and the like) or a plate. The degree of stimulatory abilityof the conjugate is influenced by the density of sugars conjugated tothe carrier (see Example 4). Preferably, the sugar molecules comprise atleast 10-25% of the conjugate by weight, more preferably at least 15-25%of the conjugate by weight and even more preferably at least 20-25% ofthe conjugate by weight. In a preferred embodiment, the stimulatory formof a compound comprising a Lewis antigen is a conjugate of multiplecarbohydrate molecules expressing a Lewis antigen and the carrierpolyacrylamide. More preferably, the polyacrylamide conjugates comprise25 to 30 (or more) sugars/conjugate, wherein the average molecularweight of the conjugate is approximately 30 kD.

The Lewis antigens present in the conjugate can be, for example,Lewis^(y), Lewis^(x), Lewis^(a) or Lewis^(b) oligosaccharides, orderivatives thereof. For stimulation of human cells, the stimulatoryagent preferably comprises Lewis^(y) oligosaccharides or derivativesthereof. Within the stimulatory agent, the Lewis antigen can be presentwithin a larger carbohydrate structure. For example, the carbohydrateportion of the stimulatory agent can be lacto-N-fucopentaose III(LNFP-III), which has the structure:{Gal(β1-4)[Fuc(α1-3)]GlcNac(β1-3)Gal(β1-4)Glc} and comprises theLewis^(x) oligosaccharide, or lacto-N-difucohexose I (LND), which hasthe structure: {Fuc(β1-2)Gal(β1-3)[Fuc(β1-4)]GlcNac(β1-3)Gal(β1-4)Glc}and comprises the Lewis^(b) oligosaccharide. Other related carbohydratescomprising Lewis antigens that are suitable for use in a stimulatoryagent of the invention will be apparent to those skilled in the art.

In addition to conjugates comprising Lewis antigen-containing sugarsdescribed above, another form of a stimulatory agent comprising a Lewisantigen is an isolated protein that naturally expresses Lewis antigensin a form suitable for stimulatory activity. One example of such aprotein is schistosome egg antigen (SEA), which expresses the Lewis^(x)oligosaccharide. Other proteins that have been reported to express Lewisantigens include tumor-associated antigens (see e.g., Pauli, B. U., etal. (1992) Trends in Glycoscience and Glycotechnology 4:405-414;Hakomori, S-I. (1989) Adv. Cancer Res. 52:257-331) and HIV gp120(Adachi, M., et al. (1988) J. Exp. Med. 167:323-331).

Stimulatory agents for use in the methods of the invention can bepurchased commercially or can be purified or synthesized by standardmethods. Conjugates of Lewis antigen-containing sugars and a carrierprotein (e.g., HSA) are available from Accurate Chemicals, Westbury,N.Y. Conjugates of Lewis antigen-containing sugars and polyacrylamideare available from GlycoTech, Rockville, Md. Schistosome egg antigen(SEA) can be purified from Schistosoma mansoni eggs as described inHarn, D. H., et al. (1984) J. Exp. Med. 159:1371-1387. Lewisantigen-containing sugars, or derivatives thereof, can be conjugated toa carrier protein or solid support (e.g., beads or a plate) by standardmethods, for example using a chemical cross-linking agent. A widevariety of bifunctional or polyfunctional cross-linking reagents, bothhomo- and heterofunctional, are known in the art and are commerciallyavailable (e.g., Pierce Chemical Co., Rockford, Ill.).

The ability of a stimulatory agent of the invention to stimulateproduction by immune cells of at least one cytokine that regulates a Th1or Th2 response can be evaluated using an in vitro culture system suchas that described in the Examples. Cells (e.g., peripheral bloodmononuclear cells) are cultured in the presence of the stimulatory agentto be evaluated (e.g., at a concentration of 100 μM for sugarconjugates) in a medium suitable for culture of the chosen cells. Aftera period of time (e.g., 24-72 hours), production of a cytokine thatregulates development of a Th1 or Th2 response is assessed bydetermining the level of the cytokine in the culture supernatant.Preferably, the cytokine assayed is IL-10. Additionally oralternatively, IL-4 and/or PGE₂ levels can be assessed. Cytokine levelsin the culture supernatant can be measured by standard methods, such asby an enzyme linked immunosorbent assay (ELISA) utilizing a monoclonalantibody that specifically binds the cytokine. An ELISA for measuringIL-10 levels is described further in Kullberg, M. C., et al. (1992) J.Immunol. 148:3264-3270. An ELISA kit for measuring PGE₂ levels iscommercially available from Advanced Magnetics, Cambridge, Mass. Theability of a stimulatory agent to stimulate cytokine production isevidenced by a higher level of cytokine (e.g., IL-10) in thesupernatants of cells cultured in the presence of the stimulatory agentcompared to the level of cytokine in the supernatant of cells culturedon the absence of the stimulatory agent.

B. Inhibitory Agents

The inhibitory agents of the invention inhibit production by cells(e.g., T cells, macrophages or human immune cells) of at least onecytokine that regulates development of a Th1 or Th2 response. Morespecifically, cytokine production by a cell that normally occurs whenthe cell encounters a stimulatory form of a Lewis antigen can beinhibited by contacting the cell with an inhibitory agent of theinvention (i.e., the inhibitory agents of the invention act as blockingagents to inhibit cytokine production that normally would result frominteraction of the cell with a stimulatory form of a Lewis antigen). Ina preferred embodiment, the inhibitory agent is an inhibitory form of acompound comprising a Lewis antigen. A “inhibitory form of a compoundcomprising a Lewis antigen” typically is one in which the carbohydratestructure is present in a monovalent, non-crosslinked form. Preferredinhibitory agents are soluble, “free” sugars comprising a Lewis antigen.The Lewis antigen expressed by the inhibitory agent can be, for example,a Lewis^(y), Lewis^(x), Lewis^(a) or Lewis^(b) oligosaccharide (a singlesubstituted core sequence or a repetitive series of substituted coresequences) or derivatives thereof. For inhibition of human cells, theinhibitory agent preferably comprises a Lewis^(y) oligosaccharide or aderivative thereof. Within the inhibitory agent, the Lewis antigen canbe present within a larger carbohydrate structure. For example, thecarbohydrate portion of the inhibitory agent can be lacto-N-fucopentaoseIII (LNFP-III), which has the structure:{Gal(β1-4)[Fuc(α1-3)]GlcNac(β1-3)Gal(β1-4)Glc} and comprises theLewis^(x) oligosaccharide, or lacto-N-difucohexose I (LND), which hasthe structure: {Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNac(β1-3)Gal(β1-4)Glc }and comprises the Lewis^(b) oligosaccharide. Other related carbohydratescomprising Lewis antigens that are suitable for use in an inhibitoryagent of the invention will be apparent to those skilled in the art.Soluble, free sugars comprising a Lewis antigen for use as inhibitoryagents can be purchased commercially or synthesized by standard methods(as described above).

In addition to free sugars comprising Lewis antigens, another form of aninhibitory agent is an antibody to a Lewis antigen. Polyclonalantibodies, or more preferably monoclonal antibodies (mAbs), thatspecifically bind a Lewis antigen (e.g., a Lewis^(y), Lewis^(x),Lewis^(a) or Lewis^(b) oligosaccharide, or derivatives thereof) can beused as inhibitory agents. Monoclonal antibodies to Lewis antigens, orderivatives thereof, are known in the art (e.g., mAbs to Le^(x), Le^(y),sialyl-Le^(x) or sialyl-Le^(y), from the Biomembrane Institute, Seattle,Wash., as described in Martensson, S., et al. (1995) Hum. Pathol.26:735-739; and mAbs to Le^(a) or sialyl-Le^(a), as described inKageshita, T., et al. (1995) Cancer Res. 55:1748-1751)

The ability of an inhibitory agent of the invention to inhibitproduction by immune cells of at least one cytokine that regulates a Th1or Th2 response that normally occurs when the immune cell encounters astimulatory form of a Lewis antigen can be evaluated using an in vitroculture system such as that described in the Examples. Cells (e.g.,peripheral blood mononuclear cells) are cultured in the presence of bothan inhibitory agent to be evaluated and a stimulatory agent as describedin the preceding section (e.g., a sugar conjugate at a concentration of100 μM) in a medium suitable for culture of the chosen cells. After aperiod of time (e.g., 24-72 hours), production of a cytokine thatregulates development of a Th1 or Th2 response (e.g., IL-10 or IL-4) isassessed by determining the level of the cytokine in the culturesupernatant as described in the preceding section. The ability of aninhibitory agent to inhibit cytokine production is evidenced by a lowerlevel of cytokine in the supernatants of cells cultured in the presenceof both the inhibitory agent and the stimulatory agent compared to thelevel of cytokine in the supernatant of cells cultured only in thepresence of the stimulatory agent.

II. Pharmaceutical Compositions

Another aspect of the invention pertains to pharmaceutical compositionsof the stimulatory or inhibitory agents of the invention. Thepharmaceutical compositions of the invention typically comprise astimulatory or inhibitory agent of the invention and a pharmaceuticallyacceptable carrier. As used herein “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. The type of carrier can beselected based upon the intended route of administration. In variousembodiments, the carrier is suitable for intravenous, intraperitoneal,subcutaneous, intramuscular, transdermal or oral administration. In apreferred embodiment, the composition is formulated such that it issuitable for intravenous administration. Pharmaceutically acceptablecarriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the pharmaceutical compositions of theinvention is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the modulators can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Depending on the route of administration, the agent may be coated in amaterial to protect it from the action of enzymes, acids and othernatural conditions which may inactivate the agent. For example, theagent can be administered to a subject in an appropriate carrier ordiluent co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27).Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

The active agent in the composition (i.e., a stimulatory or inhibitoryagent of the invention) preferably is formulated in the composition in atherapeutically effective amount. A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result, such as thedevelopment or progression of a Th2 or Th1 response to thereby influencethe therapeutic course of a particular disease state. A therapeuticallyeffective amount of an active agent may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the agent to elicit a desired response in the individual.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the agent are outweighed by thetherapeutically beneficial effects. In another embodiment, the activeagent is formulated in the composition in a prophylactically effectiveamount. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result, for example, influencing the development orprogression of either a Th2 or Th1 response for prophylactic purposes.Typically, since a prophylactic dose is used in subjects prior to or atan earlier stage of disease, the prophylactically effective amount willbe less than the therapeutically effective amount.

A non-limiting range for a therapeutically or prophylactically effectiveamounts of a stimulatory or inhibitory agent of the invention is 0.01nM-20 mM. It is to be noted that dosage values may vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

A stimulatory or inhibitory agent of the invention can be formulatedinto a pharmaceutical composition wherein the agent is the only activecompound therein. Alternatively, the pharmaceutical composition cancontain additional active compounds. For example, two or morestimulatory or inhibitory agents may be used in combination. Moreover, astimulatory or inhibitory agent of the invention can be combined withone or more other agents that have immunomodulatory properties. Forexample, a stimulatory or inhibitory agent may be combined with one ormore cytokines or adjuvants.

A pharmaceutical composition of the invention, comprising a stimulatoryor inhibitory agent of the invention, can be administered to a subjectto modulate immune responses (e.g., Th2 vs. Th1 responses) in thesubject. As used herein, the term “subject” is intended to includeliving organisms in which an immune response can be elicited, e.g.,mammals. Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof.

III. Modulation of Immune Responses

The invention provides immunomodulatory methods that can be used toinfluence the development of a Th1 or a Th2 response. In the methods ofthe invention, a cell (e.g., a human immune cell, a macrophage or a Tcell) is contacted with an agent that modulates interaction of acompound comprising a Lewis antigen with the cell such that productionby the cell of at least one cytokine that regulates development of a Th1or Th2 response is modulated (i.e., stimulated or inhibited). Themethods of the invention can be practiced either in vitro or in vivo.For practicing the method of the invention in vitro, cells can beobtained from a subject by standard methods and incubated (i.e.,cultured) in vitro with a stimulatory or inhibitory agent of theinvention to stimulate or inhibit, respectively, the production of atleast one cytokine that regulates development of a Th1 or Th2 response.For example, peripheral blood mononuclear cells (PBMCs) can be obtainedfrom a subject and isolated by density gradient centrifugation, e.g.,with Ficoll/Hypaque. Specific cell populations can be depleted orenriched using standard methods. For example, monocytes/macrophages canbe isolated by adherence on plastic. T cells or B cells can be enrichedor depleted, for example, by positive and/or negative selection usingantibodies to T cell or B cell surface markers, for example byincubating cells with a specific mouse monoclonal antibody (mAb),followed by isolation of cells that bind the mAb using anti-mouse-Igcoated magnetic beads. Monoclonal antibodies to cell surface markers arecommercially available.

When a stimulatory agent is used in vitro, resulting in stimulation ofthe production of at least one cytokine that regulates Th1 or Th2responses, the cytokine(s) can be recovered from the culture supernatantfor further use. For example, the culture supernatant, or a purifiedfraction thereof, can be applied to T cells in culture to influence thedevelopment of Th1 or Th2 cells in vitro. Alternatively, the culturesupernatant, or a purified fraction thereof, can be administered to asubject to influence the development of Th1 vs. Th2 responses in thesubject. Moreover, cells treated in vitro with either a stimulatory orinhibitory agent can be administered to a subject to influence thedevelopment of a Th1 vs. Th2 response in the subject. For administrationto a subject, it may be preferable to first remove residual agents inthe culture from the cells before administering them to the subject.This can be done for example by a Ficoll/Hypaque gradient centrifugationof the cells.

For practicing the methods of the invention in vivo, a stimulatory orinhibitory agent is administered to a subject in a pharmacologicallyacceptable carrier (as described in the previous section) in amountssufficient to modulate production of at least one cytokine thatregulates development of a Th1 or Th2 response in the subject. Apreferred route of administration for the agent is intravenous, althoughany route of administration suitable for achieving the desiredimmunomodulatory effect is contemplated by the invention.

The stimulatory methods of the invention (i.e., methods that use astimulatory agent) result in production of at least one cytokine(preferably IL-10 or IL-4) which promotes development of a Th2 responseand/or inhibits development of a Th1 response. Alternatively, theinhibitory methods of the invention (i.e., methods that use aninhibitory agent) inhibit the production of at least one cytokine(preferably IL-10 or IL-4) which promotes development of a Th2 responseand/or inhibits development of a Th1 response. Thus, to apply a methodof the invention to the treatment of a disease condition wherein a Th1response is beneficial, an immunomodulatory method is selected thatpromotes such a Th1 response (while downregulating a Th2 response).Alternatively, to apply a method of the invention to the treatment of adisease condition wherein a Th2 response is beneficial, animmunomodulatory method is selected that promotes such a Th2 response(while downregulating a Th1 response). Application of the methods of theinvention to the treatment of disease conditions may result in cure ofthe condition, a decrease in the type or number of symptoms associatedwith the condition, either in the long term or short term (i.e.,amelioration of the condition) or simply a transient beneficial effectto the subject.

Numerous disease conditions associated with a predominant Th1 orTh2-type response have been identified and could benefit from modulationof the type of response mounted in the individual suffering from thedisease condition. Application of the immunomodulatory methods of theinvention to such diseases is described in further detail below.

A. Cancer

The inhibitory methods of the invention can be used to inhibit theproduction of cytokines that upregulate Th2 responses and downregulateTh1 responses in cancer patients, thereby promoting a Th1 response inthe patients to ameliorate the course of the disease. The expression ofTh2-promoting cytokines, in particular IL-10, has been reported to beelevated in cancer patients (see e.g., Yamamura, M., et al. (1993) J.Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad.Sci. USA 89:7708-7712). Moreover, malignant cells have been reported toexpress tumor-associated antigens that comprise Lewis antigens (seee.g., Pauli, B. U., et al. (1992) Trends in Glycoscience andGlycotechnology 4:405-414; Hakomori, S-I. (1989) Adv. Cancer Res.52:257-331; Martensson, S., et al. (1995) Hum. Pathol. 26:735-739;Kageshita, T., et al. (1995) Cancer Res. 55:1748-1751; Ohta, S., et al.(1995) Immunol. Letters 44:35-40). Still further, as demonstrated hereinin Examples 2 and 3, peripheral blood mononuclear cells from patientswith a variety of cancers (e.g., lung carcinoma, colon carcinoma or Blymphoma) respond to stimulation with Lewis antigen-protein carrierconjugates by producing IL-10. Thus, in the natural setting, stimulatoryforms of Lewis antigens on tumor cells likely stimulate IL-10 productionin cancer patients, thereby shifting the immune response toward a Th2response and away from a Th1 response. This Th2 to Th1 shift can becounteracted by treating a cancer patient with an inhibitory agent ofthe invention to inhibit IL-10 production in the patient and promote aTh1 response.

A preferred inhibitory agent for inhibiting IL-10 production in humansubjects comprises an inhibitory form of a Lewis^(y) oligosaccharide orderivative thereof (e.g., soluble, free Le^(y)), although the particularLewis antigen effective for inhibiting IL-10 production in individualcancer patients may vary for different patients and/or tumors (e.g., asshown in Examples 2 and 3, cells from two patients with B lymphoma andone patient with colon carcinoma responded preferentially to Le^(y),whereas cells from one patient with B lymphoma responded preferentiallyto Le^(b) and cells from one patient with lung carcinoma responded toLe^(x)). To determine which inhibitory agent is likely to be mosteffective for a particular cancer patient, it may be necessary first todetermine which Lewis antigen is expressed on the tumor cells of thepatient (e.g., by reacting a sample of tumor cells in vitro with a panelof mAbs that bind to different Lewis antigens) and/or which Lewisantigen the cells from the cancer patient preferentially respond to invitro (e.g., by culturing PBMCs from the cancer patient in vitro withvarious stimulatory forms of Lewis antigens in an assay as described inthe Examples). Based on these in vitro tests, an appropriate inhibitoryagent can be selected that, when administered to the cancer patient,will block the stimulation of IL-10 production that results when cellsof the cancer patient encounter Lewis antigens on tumor cells in vivo.

B. Infectious Disease

The inhibitory methods of the invention also can be used to inhibit theproduction of cytokines that upregulate Th2 responses and downregulateTh1 responses in patients with infectious diseases, thereby promoting aTh1 response in the patients to ameliorate the course of the disease.The expression of Th2-promoting cytokines, in particular IL-10, has beenreported to increase during a variety of infections, including HIVinfection, tuberculosis, leishmaniasis, schistosomiasis, filarialnematode infection and intestinal nematode infection (see e.g.; Shearer,G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, Mand Shearer, G. M. (1993) Immunology Today 14:107-111; Fauci, A. S.(1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992)Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J. Exp.Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327;Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270; Bancroft, A.J., et al. (1993) J. Immunol. 150:1395-1402; Pearlman, E., et al. (1993)Infect. Immun. 61:1105-1112; Else, K. J., et al. (1994) J. Exp. Med.179:347-351) and is associated with a Th1 to Th2 shift. Infected cellsand/or proteins from infectious agents have been reported to expressLewis antigens. For example, the Lewis^(y) oligosaccharide has beenreported to be expressed by HIV-infected cells (Adachi, M., et al.(1988) J. Exp. Med. 167:323-331). Schistosome egg antigen expresses theLewis^(x) oligosaccharide (Ko, A., et al. (1987) Proc. Natl. Acad. Sci.USA 87:4159-4163). Liver cells in chronic active hepatitis express theLewis^(y) oligosaccharide (Muguruma, M, et al. (1994) Anatomic Pathology102:176-181), while Kupffer cells express sialyl oligomeric Lewis^(x)(Okado, Y. and Tsuji, T. (1990) Lancet 335:1302-1307). Thus, during thecourse of various natural infection, stimulatory forms of Lewis antigenson infected cells and/or expressed by proteins of the infectious agentlikely stimulate IL-10 production in the infected subject, therebyshifting the immune response toward a Th2 response and away from a Th1response. This Th2 to Th1 shift can be counteracted by treating theinfected subject with an inhibitory agent of the invention to inhibitIL-10 production in the infected subject and promote a Th1 response(similar to the approach described above for treating cancer patients).The inhibitory agent that is likely to be most effective for treatmentof a particular infection can be determined by approaches similar tothose described above for selecting inhibitory agents to treat cancerpatient (for example, the type of Lewis antigen expressed by infectedcells and/or proteins of the infectious agent can be determined and/orone can determine which Lewis antigen the cells from the subjectpreferentially respond to in vitro).

C. Allergies:

Allergies are mediated through IgE antibodies whose production isregulated by the activity of Th2 cells and the cytokines producedthereby. In allergic reactions, IL-4 is produced by Th2 cells, whichfurther stimulates production of IgE antibodies and activation of cellsthat mediate allergic reactions, i.e., mast cells and basophils. IL-4also plays an important role in eosinophil mediated inflammatoryreactions. As demonstrated in Example 2, T cells from a patient allergicto pollen respond to stimulation with stimulatory forms of Lewisantigens by producing IL-10. Production of IL-10 in allergic patientsmay promote a Th2 response and downregulates a Th1 response, therebyexacerbating the allergic condition. An allergic subject can be treatedwith an inhibitory agent of the invention to inhibit IL-10 production inthe subject and shift the immune response away from a Th2 response andtoward a Th1 response as a means to alleviate the allergic condition.

Allergic reactions may be systemic or local in nature, depending on theroute of entry of the allergen and the pattern of deposition of IgE onmast cells or basophils. Thus, for treatment of an allergic subject, aninhibitory agent of the invention can be administered eithersystemically or locally. Moreover, it may be beneficial to coadministerto the subject the allergen together with the inhibitory agent toinhibit (e.g., desensitize) the allergen-specific response.

D. Autoimmune Diseases:

The methods of the invention also can be used therapeutically fortreating autoimmune diseases which are associated with a Th1- orTh2-type dysfunction. Many autoimmune disorders are the result ofinappropriate activation of T cells that are reactive against selftissue and that promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. It has been shown thatmodulation of T helper-type responses can either have a beneficial ordetrimental effect on an autoimmune disease. For example, inexperimental allergic encephalomyelitis (EAE), stimulation of a Th2-typeresponse by administration of IL-4 at the time of the induction of thedisease diminishes the intensity of the autoimmune disease (Paul, W. E.,et al. (1994) Cell 76:241-251). Furthermore, recovery of the animalsfrom the disease has been shown to be associated with an increase in aTh2-type response as evidenced by an increase of Th2-specific cytokines(Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364). Moreover, Tcells that can suppress EAE secrete Th2-specific cytokines (Chen, C., etal. (1994) Immunity 1:147-154). Since stimulation of a Th2-type responsein EAE has a protective effect against the disease, stimulation of a Th2response in subjects with multiple sclerosis (for which EAE is a model)may be beneficial therapeutically.

Similarly, stimulation of a Th2-type response in type I diabetes in miceprovides a protective effect against the disease. Indeed, treatment ofNOD mice with IL-4 (which promotes a Th2 response) prevents or delaysonset of type I diabetes that normally develops in these mice (Rapoport,M. J., et al. (1993) J. Exp. Med. 178:87-99). Thus, stimulation of a Th2response in a subject suffering from or susceptible to diabetes mayameliorate the effects of the disease or inhibit the onset of thedisease.

Yet another autoimmune disease in which stimulation of a Th2-typeresponse may be beneficial is rheumatoid arthritis (RA). Studies haveshown that patients with rheumatoid arthritis have predominantly Th1cells in synovial tissue (Simon, A. K., et al., (1994) Proc. Natl. AcadSci. USA 91:8562-8566). By stimulating a Th2 response in a subject withRA, the detrimental Th1 response can be concomitantly downmodulated tothereby ameliorate the effects of the disease.

To treat an autoimmune disease in which a Th2-type response isbeneficial to the course of the disease in the subject, a stimulatoryagent of the invention can be administered to the subject in amountssufficient to stimulate a Th2-type response. The stimulatory agent canbe used alone, or in combination with one or more additional agents thatpromote Th2 responses (e.g., Th2-promoting cytokines, such as IL-4 orIL-10). Depending on the disease, the stimulatory agent may beadministered either systemically or locally. For example in the case ofrheumatoid arthritis, the agent may be administered directly into thejoints. For systemic treatment, the stimulatory agent preferably isadministered intravenously. Alternative to direct administration of thestimulatory agent to the subject, autoimmune diseases may be treated byan ex vivo approach. In this case, immune cells (e.g., T cells,macrophages and/or B cells) are obtained from a subject having anautoimmune disease, cultured in vitro with a stimulatory agent of theinvention to stimulate production by the cells of one or more cytokinesthat promote a Th2 response (e.g., IL-10), followed by readministrationof the cells to the subject.

In contrast to the autoimmune diseases described above in which a Th2response is desirable, other autoimmune diseases may be ameliorated by aTh1-type response. Such diseases can be treated using an inhibitoryagent of the invention (as described above for cancer and infectiousdiseases). The treatment may be further enhanced by administrating aTh1-promoting cytokine (e.g., IFN-γ) to the subject in amountssufficient to further stimulate a Th1-type response.

The efficacy of agents for treating autoimmune diseases can be tested inthe above described animal models of human diseases (e.g., EAE as amodel of multiple sclerosis and the NOD mice as a model for diabetes) orother well characterized animal models of human autoimmune diseases.Such animal models include the mrl/lpr/lpr mouse as a model for lupuserythematosus, murine collagen-induced arthritis as a model forrheumatoid arthritis, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856). A modulatory (i.e., stimulatory or inhibitory) agent of theinvention is administered to test animals and the course of the diseasein the test animals is then monitored by the standard methods for theparticular model being used. Effectiveness of the modulatory agent isevidenced by amelioration of the disease condition in animals treatedwith the agent as compared to untreated animals (or animals treated witha control agent).

Non-limiting examples of autoimmune diseases and disorders having anautoimmune component that may be treated according to the inventioninclude diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

E. Inflammatory Bowel Disease in Infants

The methods and compositions of the invention also can be used todownregulate inflammatory immune responses in the bowel in newborninfants. The largest biological source for Lewis-antigen containingoligosaccharides is human breast milk and breast fed babies have a lowerincidence of diarrhea and colitis/inflammatory bowel disease.Commercially available formula was tested for the presence of Lewisantigen-containing sugars by ELISA and was found not to containdetectable amounts of these sugars, nor did the ingredients list thesesugars as additives. Nonfat dry milk was listed as an additive to theformula but when non-fat dry milk was tested for the presence of Lewisantigen-containing sugars by ELISA, again none were detected. Thus,Lewis antigen-containing compounds as described herein can be added tocommercial baby formulas and administered to infants orally to inhibitdiarrhea and colitis/inflammatory bowel disease. Accordingly, theinvention provides an infant formula comprising one or more Lewisantigen-containing compounds as an additive.

In addition to the foregoing disease situations, the immunomodulatorymethods of the invention also are useful for other purposes. Forexample, the stimulatory methods of the invention (i.e., methods using astimulatory agent) can be used to stimulate production of Th2-promotingcytokines (such as IL-10 or IL-4) in vitro for commercial production ofthese cytokines (e.g., cells can be cultured with a stimulatory agent invitro to stimulate IL-10 or IL-4 production and the IL-10 or IL-4 can berecovered from the culture supernatant, further purified if necessary,and packaged for commercial use).

Furthermore, the immunomodulatory methods of the invention can beapplied to vaccinations to promote either a Th1 or a Th2 response to anantigen of interest in a subject. That is, the agents of the inventioncan serve as adjuvants to direct an immune response to a vaccine eitherto a Th1 response or a Th2 response. For example, to stimulate anantibody response to an antigen of interest (i.e., for vaccinationpurposes), the antigen and a stimulatory agent of the invention can becoadministered to a subject to promote a Th2 response to the antigen inthe subject, since Th2 responses provide efficient B cell help andpromote IgG1 production. Alternatively, to promote a cellular immuneresponse to an antigen of interest, the antigen and an inhibitory agentof the invention can be coadministered to a subject to promote a Th1response to the antigen in a subject, since Th1 responses favor thedevelopment of cell-mediated immune responses (e.g., delayedhypersensitivity responses). The antigen of interest and the modulatoryagent can be formulated together into a single pharmaceuticalcomposition or in separate compositions. In a preferred embodiment, theantigen of interest and the modulatory agent are administeredsimultaneously to the subject. Alternatively, in certain situations itmay be desirable to administer the antigen first and then the modulatoryagent or vice versa (for example, in the case of an antigen thatnaturally evokes a Th1 response, it may be beneficial to firstadminister the antigen alone to stimulate a Th1 response and thenadminister a stimulatory agent, alone or together with a boost ofantigen, to shift the immune response to a Th2 response).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLE 1

In this example, the ability of macrophages to produce IL-10 uponstimulation with various oligosaccharide conjugates was examined.Granuloma macrophages were prepared from C57BL/6 mice as described inFlores Villanueva, P. O., et al. (1994) J. Immunol. 153:5190-5199 andFlores Villanueva, P. O., et al. (1994) J. Immunol. 152:1847-1855. Twomillion cells in 1 ml of DMEM, 10% FCS were incubated with either nostimulant (i.e., medium alone; a negative control), lipopolysaccharide(LPS; at 20 μg/ml; a positive control), schistosome egg antigen (SEA; at10 μg/ml), various sugars conjugated to human serum albumin (at 100μg/ml) or human serum albumin alone (HSA; at 100 μg/ml; a negativecontrol). The sugar conjugates tested were lacto-N-fucopentaose III(LNFP-III), lacto-N-neotetraose (LNT) and Lewis^(y) (Le^(y)). Theoligosaccharide conjugates were obtained from Accurate Chemicals,Westbury, N.Y.

At various time points (24, 48 and 72 hours), the level of IL-10 in thesupernatant was determined by two-site ELISA as described by Kullberg,et al. (J. Immunol. (1982) 148:3264-3270). Briefly, polystyrenemicrotiter plates (Costar) were coated with IL-10-specific monoclonalantibodies (clone SXC-1; DNAX Corp.). Culture supernatants wereincubated on the antibody-coated plates and then probed withbiotinylated antibody. Serial dilutions of recombinant mouse IL-10(PharMingen) were assayed simultaneously to construct a standard curvefor relative IL-10 concentration. To detect the biotinylated antibodybound to IL-10, avidin-peroxidase conjugate (Sigma Chemical Corp., St.Louis, Mo.) was diluted in phosphate buffered saline (PBS)/10% fetalcalf serum and added to the wells for 30 minutes at room temperature.The plates were washed with PBS/0.5% Tween 20 (Fisher), and thentetramethylbenzidine substrate (Kirkegaard & Perry Laboratories) wasadded; the reaction was stopped with 0.4 M phosphoric acid and read at450 nm in a UvMax Reader (Molecular Devices).

The results are shown below in Table I. TABLE I IL-10 Secretion byGranuloma Macrophages IL-10 LEVEL (PG/ML) STIMULANTS 24 HR 48 HR 72 HRMedium  898 1510  871 LPS 7864 6559 4555 SEA 2805 2282 2835 HSA-LNFP-III1967 1682 1211 HSA-LNT 1038 1298  912 HSA-LeY 3860 3955 3076 HSA 12471214 1089

The greatest response was observed with the Lewis^(y) conjugate as thestimulant. A less vigorous response was seen with SEA and the LNFP-IIIconjugate, both of which expresses the Lewis^(x) antigen. No responsewas seen with the LNT conjugate. The results shown in Table I indicatethat granuloma macrophages can produce IL-10 upon stimulation withcompounds comprising a Lewis antigen, in particular a Lewis^(y) antigen.

EXAMPLE 2

In this example, the ability of peripheral blood mononuclear cells(PBMCs) from humans suffering from various disorders to produce IL-10upon stimulation with various oligosaccharide conjugates was examined.Human PBMCs were obtained from: 1) an individual who was allergic topollen; 2) an individual suffering from lung carcinoma and 3) anindividual suffering from colon carcinoma. One million PBMC in RPMI-1640medium containing 10% human AB serum were incubated with either nostimulant (i.e., medium alone; a negative control), lipopolysaccharide(LPS; at 20 μg/ml; a positive control), various sugars conjugated tohuman serum albumin (at 100 μg/ml) or human serum albumin alone (HSA; at100 μg/ml; a negative control). The sugar conjugates tested werelacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose III (LNFP-III),lacto-N-neotetraose (LNT) and Lewis^(y) (Le^(y)).

After 24 hours, the level of IL-10 (in pg/ml) in the supernatant wasdetermined by two-site ELISA as described by in Example 1, using ananti-human IL-10 monoclonal antibody obtained from PharMingen.Additionally, cellular proliferation was measured by standard³H-thymidine uptake at 90 hours.

The results are shown below in Table II. TABLE II Responses of HumanCells from Allergic or Cancer Patients to Various Sugar ConjugatesPOLLEN LUNG COLON ALLERGIC CARCINOMA CARCINOMA STIMULANT CPM IL-10 CPMIL-10 CPM IL-10 Medium 649 160 143  1200 30  0 LPS 1946 906 232  1400222 8221  HSA-LNFP-I 490 432 3452  10000 423 347 HSA-LNFP-III 1456 442137  1000 270 202 HSA-LNT 1133 220 141  1000 186 304 HSA-LeY 5960 736458  1200 934 689 HSA 460 166 152  1200 141  0

The greatest response by cells of the pollen allergic individual wasobserved with the Lewis^(y) conjugate as the stimulant. Less vigorousresponses were seen with the LNFP-I and LNFP-III conjugates. A minimalresponse was seen with the LNT conjugate. The greatest response by cellsof the individual suffering from colon carcinoma also was observed withthe Lewis^(y) conjugate as the stimulant. Less vigorous responses wereseen with the LNFP-I, LNFP-III and LNT conjugates. For the individualwith lung carcinoma, a vigorous response was observed only with theLNFP-I conjugate. The results shown in Table II indicate that humanPBMCs from individuals suffering from various disorders (e.g., allergiesor cancers) can produce IL-10 upon stimulation with compounds comprisinga Lewis antigen.

EXAMPLE 3

In this example, the ability of peripheral blood mononuclear cells(PBMCs) from three humans with B lymphomas to produce IL-10 uponstimulation with various oligosaccharide conjugates was examined. Onemillion PBMC in RPMI-1640 medium containing 10% human AB serum wereincubated with either no stimulant (i.e., medium alone; a negativecontrol), lipopolysaccharide (LPS; at 20 μg/ml; a positive control),various sugars conjugated to human serum albumin (at 100 μg/ml) or humanserum albumin alone (HSA; at 100 μg/ml; a negative control). The sugarconjugates tested were lacto-N-fucopentaose I (LNFP-I),lacto-N-fucopentaose III (LNFP-III), lacto-N-neotetraose (LNT),Lewis^(y) (Le^(y)) and lacto-N-difucohexose I (LND).

After 24 hours, the level of IL-10 (in pg/ml) in the supernatant wasdetermined by two-site ELISA as described by in Example 2. Additionally,cellular proliferation was measured by standard ³H-thymidine uptake at90 hours.

The results are shown below in Table III. TABLE III Responses of HumanPBMC from B Lymphoma Patients to Various Sugar Conjugates B- B-LYM-B-LYMPHOMA-1 LYMPHOMA-2 PHOMA-3 STIMULANT CPM IL-10 CPM IL-10 CPM IL-10Medium 444  814 288  0 1710  0 LPS 80424 6053 1084 201 3424 1049 HSA-LNFP-I 889 2422 970  0 4504  0 HSA-LNFP-III 459 4386 1009  0 3425 64 HSA-LNT 444 1081 731  0 3530  39 HSA-LeY 6211 6786 1099  2 5838 210HSA-LND 1214 3521 502 307 4324  0 HSA 248  945 660  0 3149  0

The greatest response by cells of the B-lymphoma-1 patient was observedwith the Lewis^(y) conjugate as the stimulant. Less vigorous responseswere seen with the LNFP-I, LNFP-III and LND conjugates. Similarly, thegreatest response by cells of the B-lymphoma-3 patient was observed withthe Lewis^(y) conjugate as the stimulant. Less vigorous responses wereseen with the LNFP-III and LNT conjugates. For the B-lymphoma-2 patient,a vigorous response was observed only with the LND conjugate, whichexpresses the Lewis^(b) oligosaccharide. The results shown in Table IIIindicate that human PBMCs from individuals suffering from B lymphomascan produce IL-10 upon stimulation with compounds comprising a Lewisantigen.

EXAMPLE 4

In this example, the ability of splenocytes from uninfected mice or miceinfected with S. mansoni to produce IL-10 or IL-4 was examined uponstimulation of the cells with various oligosaccharide conjugates havingdifferent sugar densities. In particular, the stimulatory ability ofLNFP III-HSA (which comprises the Lewis^(x) antigen) was compared toconjugates of Lewis^(a) antigen, Lewis^(x) antigen, sialyl-Lewis^(x)antigen or sialyl-Lewis^(a) antigen to a polyacrylamide (PAA) carriermolecule. The PAA conjugates (obtained from GlycoTech, Rockville, Md.)have a ligand density approximately 5-6 fold higher than that of the HSAconjugate. Within the LNFP III-HSA conjugate, approximately 10-12 sugarmolecules/HSA molecule are present. The total conjugate weight is 67 kD,of which the LNFP III molecules comprise approximately 3.5-4.0% of thetotal weight. Thus, when 50 μg/ml of LNFP III-HSA conjugate is used forstimulation, the actual concentration of sugar used is between 1.5 and2.0 μg/ml. Within the polyacrylamide conjugates, approximately 25-30sugar molecules/PAA molecule are present. The total conjugate isapproximately 30 kD, of which the Lewis antigens comprise approximately20-24% of the total weight. Thus, when 50 μg/ml of Lewis antigen-PAAconjugate is used for stimulation, the actual concentration of sugarused is approximately 10-12 μg/ml.

In these experiments unfractionated splenocytes isolated from 7-week S.mansoni-infected CBA/J mice or uninfected mice were used. Thesplenocytes were incubated either with media alone, Con A (50 μg/ml),LPS (50 μg/ml), anti-IgM (25 μg/ml) (all as controls), SEA (5-10 μg/ml),HSA-LNFP III (50 μg/ml), Lewis^(a) -PAA (2, 10 or 50 μg/ml),Lewis^(x)-PAA (2, 10 or 50 μg/ml), sialyl-Lewis^(x)-PAA (2, 10 or 50μg/ml) or sialyl-Lewis^(a)-PAA (2, 10 or 50 μg/ml).

Proliferation of the splenocytes was assessed by standard tritiatedthymidine uptake. The results are shown in FIGS. 1A, 1B, 1C and 2D.Lewis^(a), Lewis^(x), sialyl-Lewis^(x) and sialyl-Lewis^(a), eachconjugated to PAA, did not stimulate proliferation of the splenocytesfrom either infected or uninfected mice.

The level of IL-10 production was assessed as described in Example 1.The results are shown in FIGS. 2A, 2B, 2C and 2D. Lewis^(a), Lewis^(x),sialyl-Lewis^(x) and sialyl-Lewis^(a), each conjugated to PAA, were ableto stimulate IL-10 production by splenocytes from infected mice and weresubstantially more effective at stimulating IL-10 production than LNFPIII-HSA (compare FIG. 2A and FIG. 2B). For example, 50 μg/ml of LNFPIII-HSA stimulated the production of approximately 1000 pg/ml of IL-10(see FIG. 2A), whereas the same amount of Lewis^(x)-PAA andsialyl-Lewis^(a)-PAA stimulated production of approximately 17,500 pg/mlof IL-10 and the same amount of Lewis^(a)-PAA and sialyl-Lewis^(x)-PAAstimulated production of approximately 7,500 pg/ml of IL-10 (see FIG.2B). Lewis^(x)-PAA was also able to stimulated IL-10 production bysplenocytes from uninfected mice (e.g., production of approximately 600pg/ml of IL-10 was stimulated by 50 μg/ml of Lewis^(x)-PAA, whereas thesame amount of LNFP III-HSA did not stimulate IL-10 production bysplenocytes from uninfected mice; compare FIGS. 2C and 2D).

The level of IL-4 production in the cultures was assessed by a standardELISA using an anti-mouse IL-4 monoclonal antibody obtained fromPharMingen. The results are shown in FIGS. 3A, 3B, 3C and 3D. TheLewis^(x)-PAA was effective at stimulating IL-4 production bysplenocytes from both uninfected and infected mice (see FIGS. 3B and3D). Sialyl-Lewis^(a)-PAA stimulated lower amounts of IL-4 production insplenocytes from infected mice (see FIG. 3B), as did LNFP III-HSA (seeFIG. 3A), whereas these conjugates did not stimulate IL-4 production bysplenocytes from uninfected mice (see FIGS. 3C and 3D).

These results demonstrate that Lewis antigen-containing sugarconjugates, in particular those with a high sugar density, can stimulateIL-10 and IL-4 production by naive splenocytes (i.e., splenocytes fromuninfected mice). In particular, IL-4 production was greater bystimulated naive splenocytes than stimulated splenocytes from infectedmice. This IL-4 production likely is not by B cells. Rather this IL-4production is likely by T cells and/or other non-T, non-B cells (e.g.,mast cells, basophils and/or eosinophils).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An immunomodulatory method comprising contacting a human immune cellwith an agent that modulates interaction of a compound comprising aLewis antigen with the human immune cell such that production by thehuman immune cell of at least one cytokine that regulates development ofa T helper type 1 (Th1) or T helper type 2 (Th2) response is modulated.2. The method of claim 1, wherein production of IL-10 by the humanimmune cell is stimulated.
 3. The method of claim 2, wherein the agentis a stimulatory form of a compound comprising a Lewis antigen.
 4. Themethod of claim 3, wherein the agent comprises a Lewis^(y)oligosaccharide or a derivative thereof.
 5. The method of claim 3,wherein the agent comprises a Lewis^(x) oligosaccharide or a derivativethereof.
 6. The method of claim 3, wherein the agent comprises aLewis^(a) oligosaccharide or a derivative thereof, or a Lewis^(b)oligosaccharide or a derivative thereof.
 7. The method of claim 3,wherein the agent is administered to a human subject such thatproduction by human immune cells of the human subject of at least onecytokine that regulates development of a Th1 or Th2 response isstimulated.
 8. The method of claim 1, wherein production of IL-10 by thehuman immune cell is inhibited.
 9. The method of claim 8, wherein theagent is an inhibitory form of a compound comprising a Lewis antigen.10. The method of claim 9, wherein the agent comprises a Lewis^(y)oligosaccharide or a derivative thereof.
 11. The method of claim 9,wherein the agent comprises a Lewis^(x) oligosaccharide or a derivativethereof.
 12. The method of claim 9, wherein the agent comprises aLewis^(a) oligosaccharide or a derivative thereof, or a Lewis^(b)oligosaccharide or a derivative thereof.
 13. The method of claim 9,wherein the agent is administered to a human subject such thatproduction by human immune cells of the human subject of at least onecytokine that regulates development of a Th1 or Th2 response isinhibited.
 14. The method of claim 1, wherein the human immune cell is aT cell.
 15. The method of claim 2, wherein the human immune cell is amacrophage.
 16. The method of claim 3, wherein the human immune cell isa B cell.
 17. An immunomodulatory method comprising contacting amacrophage with an agent that modulates interaction of compoundcomprising a Lewis antigen with the macrophage such that production bythe macrophage of at least one cytokine that regulates development of aT helper type 1 (Th1) or T helper type 2 (Th2) response is modulated.18. The method of claim 17, wherein production of IL-10 by themacrophage is stimulated.
 19. The method of claim 18, wherein the agentis a stimulatory form of a compound comprising a Lewis antigen.
 20. Themethod of claim 19, wherein the agent comprises a Lewis^(y)oligosaccharide or a derivative thereof.
 21. The method of claim 19,wherein the agent comprises a Lewis^(x) oligosaccharide or a derivativethereof.
 22. The method of claim 19, wherein the agent comprises aLewis^(a) oligosaccharide or a derivative thereof, or a Lewis^(b)oligosaccharide or a derivative thereof.
 23. The method of claim 19,wherein the agent is administered to a subject such that production bymacrophages of the subject of at least one cytokine that regulatesdevelopment of a Th1 or Th2 response is stimulated.
 24. The method ofclaim 17, wherein production of IL-10 by the macrophage is inhibited.25. The method of claim 24, wherein the agent is an inhibitory form of acompound comprising a Lewis antigen.
 26. The method of claim 25, whereinthe agent comprises a Lewis^(y) oligosaccharide or a derivative thereof.27. The method of claim 25, wherein the agent comprises a Lewis^(x)oligosaccharide or a derivative thereof.
 28. The method of claim 25,wherein the agent comprises a Lewis^(a) oligosaccharide or a derivativethereof, or a Lewis^(b) oligosaccharide or a derivative thereof.
 29. Themethod of claim 25, wherein the agent is administered to a subject suchthat production by macrophages of the subject of at least one cytokinethat regulates development of a Th1 or Th2 response is inhibited.
 30. Animmunomodulatory method comprising contacting a T cell with an agentthat modulates interaction of a compound comprising a Lewis antigen witha T cell such that production by the T cell of at least one cytokinethat regulates development of a T helper type 1 (Th1) or T helper type 2(Th2) response is modulated.
 31. The method of claim 30, whereinproduction of IL-10 by the T cell is stimulated.
 32. The method of claim31, wherein the agent is a stimulatory form of a compound comprising aLewis antigen.
 33. The method of claim 32, wherein the agent comprises aLewis^(y) oligosaccharide or a derivative thereof.
 34. The method ofclaim 32, wherein the agent comprises a Lewis^(x) oligosaccharide or aderivative thereof.
 35. The method of claim 32, wherein the agentcomprises a Lewis^(a) oligosaccharide or a derivative thereof, or aLewis^(b) oligosaccharide or a derivative thereof.
 36. The method ofclaim 32, wherein the agent is administered to a subject such thatproduction by T cells of the subject of at least one cytokine thatregulates development of a Th1 or Th2 response is stimulated.
 37. Themethod of claim 30, wherein production of IL-10 by the T cell isinhibited.
 38. The method of claim 37, wherein the agent is aninhibitory form of a compound comprising a Lewis antigen.
 39. The methodof claim 38, wherein the agent comprises a Lewis^(y) oligosaccharide ora derivative thereof.
 40. The method of claim 38, wherein the agentcomprises a Lewis^(x) oligosaccharide or a derivative thereof.
 41. Themethod of claim 29, wherein the agent comprises a Lewis^(a)oligosaccharide or a derivative thereof, or a Lewis^(b) oligosaccharideor a derivative thereof.
 42. The method of claim 38, wherein the agentis administered to a subject such that production by T cells of thesubject of at least one cytokine that regulates development of a Th1 orTh2 response is inhibited.
 43. A pharmaceutical composition comprisingan agent that modulates interaction of a compound comprising a Lewisantigen with human immune cells of a human subject in an amountsufficient to modulate production by the human immune cells of at leastone cytokine that regulates development of a T helper type 1 (Th 1) or aT helper type 2 (Th2) response when the agent is administered to thehuman subject, and a pharmaceutically acceptable carrier.
 44. Thepharmaceutical composition of claim 43, wherein the agent is astimulatory form of a compound comprising a Lewis antigen.
 45. Thepharmaceutical composition of claim 44, wherein the agent comprises aLewis^(y) oligosaccharide or a derivative thereof.
 46. Thepharmaceutical composition of claim 44, wherein the agent comprises aLewis^(x) oligosaccharide or a derivative thereof.
 47. Thepharmaceutical composition of claim 44, wherein the agent comprises aLewis^(a) oligosaccharide or a derivative thereof, or a Lewis^(b)oligosaccharide or a derivative thereof.
 48. The pharmaceuticalcomposition of claim 43, wherein the agent is an inhibitory form of acompound comprising a Lewis antigen.
 49. The pharmaceutical compositionof claim 48, wherein the agent comprises a Lewis^(y) oligosaccharide ora derivative thereof.
 50. The pharmaceutical composition of claim 48,wherein the agent comprises a Lewis^(x) oligosaccharide or a derivativethereof.
 51. The pharmaceutical composition of claim 48, wherein theagent comprises a Lewis^(a) oligosaccharide or a derivative thereof, ora Lewis^(b) oligosaccharide or a derivative thereof.
 52. Apharmaceutical composition comprising an agent that modulatesinteraction of a compound comprising a Lewis antigen with macrophages ofa subject in an amount sufficient to modulate production by themacrophages of at least one cytokine that regulates development of a Thelper type 1 (Th1) or a T helper type 2 (Th2) response when the agentis administered to the subject, and a pharmaceutically acceptablecarrier.
 53. A pharmaceutical composition comprising an agent thatmodulates interaction of a compound comprising a Lewis antigen with Tcells of a subject in an amount sufficient to modulate production by theT cells of at least one cytokine that regulates development of a Thelper type 1 (Th1) or a T helper type 2 (Th2) response when the agentis administered to the subject, and a pharmaceutically acceptablecarrier.
 54. A pharmaceutical composition comprising an agent comprisinga Lewis^(y) antigen, or derivative thereof, in an amount sufficient tomodulate production of at least one cytokine that regulates developmentof a T helper type 1 (Th1) or a T helper type 2 (Th2) response when theagent is administered to the subject, and a pharmaceutically acceptablecarrier.
 55. A pharmaceutical composition comprising a conjugate of aLewis antigen, or derivative thereof, and a carrier molecule, such thatthe Lewis antigen, or derivative thereof, comprises 10-25% of theconjugate by weight, in an amount sufficient to stimulate production ofat least one cytokine that regulates development of a T helper type 1(Th1) or a T helper type 2 (Th2) response when the agent is administeredto the subject, and a pharmaceutically acceptable carrier.